Over 5 million Americans are diagnosed with heart failure yearly. Heart failure is generally accompanied by pathologic growth of the heart. Calcineurin signaling is an essential regulator of the hypertrophic response. A new non-hypertrophic role for calcineurin is emerging, in which calcineurin might directly maintain viability and cardiac performance, as mice with cardiac-specific calcineurin deletion have normal cardiac morphology but poor left ventricular function and premature death. To date the direct versus compensatory effects of calcineurin deletion on cardiac function remain unresolved, and reports show both positive and negative functional outcomes in calcineurin-deleted cardiac muscle. Substantial alterations in Ca2+ handling gene expression were identified in a screen of calcineurin deficient hearts. This suggests that calcineurin may regulate Ca2+ cycling proteins, which are critical functional components, as fluctuations in cytosolic Ca2+ initiate contraction and relaxation. Thus, this proposal seeks to test the hypothesis that calcineurin is directly regulating hypertrophy-independent cardiac viability and ventricular performance through post-translational and NFAT-dependent regulation of Ca2+ handling. This grant features a comparative analysis of acute gene transfer of calcineurin-targeted siRNA to cardiac myocytes and a conditional Lox-P targeted calcineurin mouse model to elucidate the direct versus indirect effects of calcineurin loss on cardiac myocyte function in vitro (aiml), and to determine the phosphorylation status of Ca2+ handling proteins (aim 2). Myocyte contractility will be examined in isolated cardiac myocytes from both genetic models using high fidelity cell shortening and Ca2+ measurements, while candidate protein and proteomic analysis will determine the phosphorylation status of Ca2+ handling proteins. A genetic rescue strategy will also be used to investigate if NFAT-dependent transcription underlies the poor survivability in calcineurin deficient mice (aim 3). This proposal should make substantial contributions to elucidating calcineurin's additional regulatory roles in the heart thereby laying the ground work for developing new therapeutic strategies for heart failure. Lay Language: Calcineurin-dependent pathologic growth of the heart is associated with end-stage heart failure. A new growth-independent regulatory role for calcineurin in cardiac function is now emerging that suggests calcineurin deletion can directly alter cardiac muscle function. This proposal is designed to elucidate the direct versus indirect effects of calcineurin on cardiac function independent of cardiac growth at the cellular and organismal level. The outcome of this work should make significant inroads into designing molecular heart failure therapies with translational potential.